The present invention is concerned with a test carrier for the analytical determination of a component of a liquid sample, especially of a body fluid, comprising a carrier layer, which is partly covered by a liquid-absorbing layer which contains a sample application zone and a transport zone, and a reagent layer which is in contact with the liquid-absorbing layer. The present invention is also concerned with the use of such a test carrier for the determination of a component of a liquid sample, especially of a body fluid.
Test carriers have achieved considerable importance in the analysis of liquids. In clinical chemistry, which involves the qualitative and quantitative analytical determination of components of body fluids, especially of blood and urine, the advantages thereof are especially appreciated. While the classical methods of working with liquid reagents require a plurality of handling steps, analytical determinations with the help of test carriers are characterized by extremely simple handling. In the analysis of urine, for example, test carriers are generally used in the form of test strips which are briefly dipped into the sample and thereafter evaluated either visually or with the use of appropriate apparatus. In the case of blood analysis, a drop of serum or, using especially advanced types of test carriers, a drop of blood is applied to a predetermined point of the test carrier.
The reaction of the sample liquid with the reagents present on the test carrier leads to a perceptible change of the test carrier and usually a change of color, brought about by the formation or liberation of a colored material. The reaction can also lead to the formation or change of a fluorescing substance or of a substance which can be perceived optically or in some other way. Test carriers for the determination of blood components are generally evaluated with the use of appropriate apparatus.
In the initial period of development of test carriers, these only possessed a single test layer upon which various reagents were combined in such a manner that even complicated reaction sequences were possible. However, single-layer test carriers do not permit chronologically defined courses of several reactions connected in sequence. This makes dependable quantitative determination of an analyte using kinetic measurements impossible. Furthermore, the stability of mixtures of different reagents is, in comparison with pure materials, often considerably reduced. This can also result in unreliable measurement values being obtained.
These considerations led to the development of test carriers in which reagents are present in different reagent layers and these can, furthermore, be arranged on a test layer in such a manner that a chronologically defined course of successively connected reactions is possible.
Federal Republic of Germany Patent Specification No. 31 30 749 describes test carriers for the determination of various parameters in serum, plasma or whole blood in which the carrier layer is formed by a longitudinal base film, upon which a liquid-absorbing layer is arranged. A rectangular covering film is fixed with one edge to the base film, which is just as wide as the base film but considerably shorter. The fixing point is in the region of the end of the liquid-absorbing layer. On the one side of the covering film facing the base foil a reagent layer, such as a reagent film is presented. The covering film with the reagent film forms a flap, the fixing thereof on to the base film thereby being such that the flap, in the resting state, does not touch the liquid-absorbing layer but can be brought into contact therewith by external manipulation. It is possible, using such a device to divide up the chronological course of a determination reaction into steps. For this purpose, the sample is first applied to a sample application zone on the liquid-absorbing layer which, when whole blood is used as sample, must be such that the erythrocytes are separated off and held back, and the erythrocyte free sample is sucked into the subsequent transport zone of the liquid-absorbing layer. The sample can then, if this is necessary, be tempered within the liquid-absorbing layer. Such is necessary, e.g., in enzymatic determinations. At a definite point of time, the covering film with its reagent layer is pressed down on to the base film and thus brought into contact with the liquid-absorbing layer. By means of this full-faced contact, the sample contained in the liquid-absorbing layer passes into the reagent layer of the flap and there, at a definite point of time, initiates a reaction which results in a detectable signal, such as a color change.
It is an important disadvantage of such test carriers that chronologically decoupled reactions in the sense of chemical, enzymatic or immunological reaction steps of a total reaction sequence necessary for the detection of a component material of a body fluid cannot be carried out. This is because as soon as the reagent layer arranged on the flap contacts the liquid absorbing layer, all of the reagents react with all of the sample components, rendering it impossible to carry out a chronological series of reactions. Further, treatment of the sample so as to condition it chemically, enzymatically or immunologically prior to the actual reaction to be measured, is not possible.
Admittedly, the reagents required for the total reaction sequence or for the conditioning can, depending upon the corresponding partial reactions, be divided into several reagent layers which, independently of one another, can be successively pressed on to the base film. Carrying out these steps to determine an analyte in a body fluid using an apparatus is extremely laborious and expensive. Furthermore, each reagent layer requires a definite volume of liquid for wetting. The more reagent layers used, the more sample volume necessary for wetting all of the layers. This is a considerable disadvantage since it is desired that only the smallest possible amounts of body fluids, for example blood, be used for the investigations.
Chronologically decoupled reactions are, in the case of the test carriers described in Federal Republic of Germany Patent Specification No. 31 30 749, in principle also possible by impregnating the liquid-absorbing layer with appropriate soluble reagents. However, this possibility suffers from the deficiency that, because of the liquid transport within the liquid-absorbing layer, impregnated reagents become enriched in the liquid front and chromatographic effects can appear which result in the formation of spatial concentration gradients and, in the end, in non-reproducible and falsified measurement results. In addition, not every material useful in a liquid absorption layer is suitable for impregnation with reagents.
Furthermore, since the liquid-absorbing layer contacts erythrocytes when the sample is whole blood, those reagents which undergo exchange reactions with erythrocytes and which disturb the detection reaction, cannot be used for the impregnation of this layer. Thus, for example, the use of materials for the impregnation of the liquid-absorbing layer which act hemolytically result in lysing of the red blood corpuscles and release of their components which can considerably disturb the test, for example due to their inherent color or due to their chemical, enzymatic or immunological reactivity. Under certain circumstances, for example in the case of the determination of bilirubin in whole blood, because of the liberation of hemoglobin from the erythrocytes, completely false measurement values are obtained.
Similar test carriers are also known which contain layers impregnated with reagents in the sample application zone, over the liquid-absorbing layer but in absorbent contact with this and possibly under an erythrocyte-separating layer, as shown in FIG. 3. Such a device is described in U.S. Pat. No. 4,477,575 and DE OS 33 23 973 (U.S. Ser. No. 77,003). In the case of the application of the sample, the materials impregnated in the reagent layer also come into contact with erythrocytes and products which result from this exchange action can pass into the transport zone of the liquid-absorbing layer where, as already stated, they can then disturb the desired reaction considerably. Therefore, the same limitations described supra apply here as well.
The last-described test carriers also possess a disadvantage in that, depending upon the nature and manner in which the sample is applied to the sample application zone, the reagent layers can be wetted by the sample to differing extents. Thus, for example, in the case of a given size of the sample application zone, the same sample volumes can be applied so that only a small surface of these reagent layers come into contact with the sample. It is also possible, however, to carry out the sample application so that the reagent layers are completely wetted by the sample. The extent of the wetting depends, therefore, upon the dexterity of the person applying the sample. The concentration of the reagent which dissolves in the sample during the wetting of the reagent layer present in the sample application zone is thus also dependent upon this parameter. As a result, although the control of reagent in the sample are supposed to be exactly adjusted, in fact inexact, greatly scattered measurements and completely false results occur.
These problems indicate, therefore, that there is a need for test carriers which only require a small amount of sample, which permit the analysis of blood without the danger of exchange actions with erythrocytes, such as hemolysis, in which the concentration of reagents in the sample are controlled and exactly adjusted and with which reactions can be chronologically decoupled.